The α-synuclein PET tracer [18F] ACI-12589 distinguishes multiple system atrophy from other neurodegenerative diseases

A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson’s disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.


Preparation of human brain homogenates from an AD donor
The procedure used was adapted from Bagchi et al., 2013, 1 describing extraction of an insoluble fraction containing protein aggregates from human brain tissue for in vitro binding and competition studies.Frozen brain tissue samples were purchased from Tissue Solutions.
Frozen tissue from the frontal cortex brain region of an AD donor with confirmed burden of Tau and b-Amyloidaggregates was used.The tissue was homogenized in high salt buffer (50mM Tris-HCl pH 7.5, 0.75M NaCl, 5mM EDTA) supplemented with protease inhibitors (Complete; Roche 11697498001) at 4°C using a glass Dounce homogenizer.The homogenate was transferred into polycarbonate centrifuge bottles (16 x 76mm; Beckman 355603) and centrifuged at 100,000 x g (38,000 RPM) in an ultracentrifuge (Beckman, XL100K) for 60 minutes at 4°C using a pre-cooled 70.1 rotor (Beckman, 342184).Pellets were resuspended in high salt buffer supplemented with 1% Triton X-100 and homogenized at 4°C.The homogenates were centrifuged again at 100,000 x g (38,000 RPM, 70.1 Ti rotor) for 60 minutes at 4°C.Pellets were resuspended in high salt buffer supplemented with 1% Triton X-100 and 1M sucrose at 4°C.The homogenates were centrifuged at 100,000 x g (38,000 RPM, 70.1 Ti rotor) for 60 minutes at 4°C.The resulting pellets containing b-Amyloid and Tau aggregates were resuspended in PBS, aliquoted and stored at -80°C until use.

Preparation of human brain homogenates from a healthy control donor
Frozen human tissue sample from the thalamus brain region, previously reported to display increased levels of MAO-B expression (Tong et al., 2013)  2 , was purchased from Tissue Solutions.Approximately 2g of frozen tissue from the thalamus brain region of a healthy control donor was used.The tissue was homogenized in high salt buffer (25mM Tris-HCl pH 7.5, 0.15M NaCl, 1mM EDTA, 1mM EGTA, 30mM NaF, Na3VO4 0.2mM, Okadaic acid 1nM, Na4P2O7 5mM,1mM PMSF) supplemented with protease inhibitors (Complete; Roche 11697498001) at 4°C using a glass Dounce homogenizer.The presence of MAO-B in the homogenates was confirmed by western blot and the absence of pathological a-syn aggregates by Alphalisa.

Radiobinding assay using [ 3 H]ACI-12589 for determination of the dissociation constant (Kd) on AD brain homogenates
To determine the dissociation constant (Kd) of [ 3 H]ACI-12589 in AD brain homogenates, the AD insoluble fraction, prepared as described above, was incubated with [ 3 H]ACI-12589 or [ 3 H]PiB at different concentrations, ranging from 2nM to 400nM or 2nM to 50nM, respectively.The reaction was performed in assay buffer (50mM Tris pH 7.5 in 0.9% NaCl, 0.1% BSA) and incubated for two hours at RT.Samples in duplicate were then filtered under vacuum in GF/C filter plates (PerkinElmer) to trap the aggregates with the bound radioligand and washed five times with ice-cold 50mM Tris pH7.5.The GF/C filters were then dried and scintillation liquid (UltimateGold, PerkinElmer) was added in each well.The filters were analyzed on a Microbeta2 scintillation counter (PerkinElmer).Co-incubation with nonradiolabeled ACI-12589 or PiB at 1µM was used to determine the level of non-specific binding.Specific binding was calculated by subtracting the non-specific signal from the total signal.Kd values were calculated by nonlinear regression, using a one site specific binding model using GraphPad Prism v7.

Radiobinding assay using [ 3 H]L-deprenyl for determination of the inhibition constant (Ki) on brain homogenates containing MAO-B
Healthy control brain homogenates containing MAO-B, prepared as described above, were incubated with a tritiated MAO-B inhibitor ([ 3 H]L-deprenyl) at 10nM and increasing concentrations of selected non-radiolabelled compounds in the range of 400pM to 2µM for two hours at RT.The reaction was performed in assay buffer (50mM Tris pH 7.5 in 0.9% NaCl, 0.1% BSA) and incubated for two hours at RT.Samples in duplicate were then filtered under vacuum in GF/C filter plates (PerkinElmer) to trap the aggregates with the bound radioligand and washed five times with ice-cold 50mM Tris pH7.5.The GF/C filters were then dried and scintillation liquid (UltimateGold, PerkinElmer) was added in each well.The filters were analyzed on a Microbeta2 scintillation counter (PerkinElmer).Non-specific signal was determined with an excess of non-radiolabelled reference ligand (2µM) and specific binding was calculated by subtracting the non-specific signal from the total signal.Competition was calculated as percent, where 0% was defined as the specific binding in the presence of vehicle and 100% as the values obtained in the presence of excess of the non-radiolabelled reference ligand.Ki values were calculated in GraphPad Prism v7 by applying a nonlinear regression curve fit using a one site, specific binding model using GraphPad Prism v7.Independent measurements were performed with two replicates.

Kinetic analyses:
Measured radioactivity concentration in whole blood and plasma were interpolated to a two-second time grid.After preliminary analysis of several models for the parent fraction, the measured parent fraction values were fitted using () = ( +   ) " , where  is the parent fraction and  and  are free model parameters 3 as this model provided adequate description of the measured data.The parent fraction model fits were subsequently multiplied to the plasma curve to generate metabolite corrected arterial input functions.The input functions were used to apply several pharmacokinetic models to the time-activity curves (one-and twotissue compartment model (1-2TCM), Logan graphical analysis and Ichise Multilinear Graphical Analysis (MA1)).
The 1TCM could not adequately describe the measured TACs.The 2TCM resulted in satisfactory model fits and systemically lower Akaike Information Criteria scores than the 1TCM, but for 26 of the total 494 regional TACs, the model parameters were not properly identified (typically k4 approached 0, resulting in unrealistically high values of distribution volume (VT)).Logan and MA1 could both fit all of the evaluated TACs and provided VT estimates that were both in high agreement with each other (0.6±3.2% difference), and in good correspondence with VT from 2TCM in all brain regions for which 2TCM provided reasonable VT estimates.For this reason, the Logan plot was selected as the model of choice.
The simplified reference tissue model and the Logan graphical analysis with reference tissue were also evaluated.As reference tissue, the occipital and cerebellar cortices were used.Binding potential with respect to non-displaceable uptake (BPND) derived either using SRTM or Logan ref correlated with both distribution volume ratios derived using the respective VT values, and with the actual VT from Logan plot (Fig. 3d).Finally, to facilitate simplified acquisition protocols, SUV ratios were calculated for three different time windows (30-50, 50-70, and 60-90-min post radioligand injection), and compared to other outcome referenceregion based outcome measures.Good correspondence was observed for all time windows, and 60-90 min post injection was selected for subsequent clinical scans.and flushed with air.The C18-cartridge was washed with 10 mL of H2O and then eluted using 2.0 mL of 50% EtOH in H2O into a transfer vial containing 10 mL NaCl solution (9 mg/mL).The C18-cartridge was then rinsed with 6 mL of NaCl solution (9 mg/mL) into the transfer vial and the complete content was subsequently transferred to the final product vial via a sterile filter (Cathivex-GV 0.22 µm, Merck Millipore).The automated radiosynthesis manufacturing process was fully validated and achieved an average of 9,3 ± 4,0 GBq of radioactive product per batch with a radiochemical yield of 25.3 ± 4.5% (n = 25).
Chromeleon version 7 (ThermoScientific) was used as chromatography system.The analytical HPLC was used for the determination of identity of product, radiochemical purity (RCP), specific radioactivity (AS), amount of ACI-12589 and unrelated UV-active impurities.
New batches of precursor also required a test synthesis with full quality control plus determination of enantiomeric purity by a chiral HPLC method.Analytical parameters used are listed in Supplementary Table 3.
TLC was used for analysis of free [ 18 F]fluoride in the product by spotting 2 µL of product onto a silica TLC-plate (TLC Silica gel 60 F₂₅₄ Aluminium TLC plate, Merck) which was then eluted using 80% MeCN in H2O.The plates were analysed using a radio-TLC scanner (detector FC-3600, Mini-Scan B-MS-1000, flowcount BioScan FC-1000, Eckert & Ziegler) connected to Dionex Universal Chromatography Interface (UCI-50/100).
The product was analysed with respect to pH, Bu4 + -content, residual solvents, endotoxin, sterility, radionuclide identity and radionuclidic purity according to European Pharmacopoeia (Ph.Eur.).Determination of fraction of intact [ 18 F]ACI-12589 in plasma was carried out for samples at baseline, 4.5, 6, 10, 20, 30, 60 and 90 min as follows.Plasma samples were processed by MeCN precipitation, treating 1 mL of plasma with 1 mL of MeCN.After vortex mixing and centrifugation at 3000 x g (4400 rpm) for 10 min, 1 mL of the supernatant was diluted with 1 mL Milli-Q water and analyzed by HPLC (pump, injector, UV-detector, and fraction collector: Ultimate 3000, Thermo Scientific; BGO radioactivity detector: Bioscan).The sample was injected on a Phenomenex Luna 10 µm C18(2) 100Å (10 x 250 mm) and eluted with a mobile phase of 60% MeOH in H2O containing 0.2% of triethylamine at a flow rate of 4 mL/min.The eluent from the column was collected in 15 different fractions (4 mL/fraction), each fraction was measured in a g-counter to determine the percentage of intact parent compound.The percentage of parent compound was calculated by dividing the radioactivity from the fractions representing parent compound by the total sum of all fractions (Supplementary Figure 3).

Blood sampling and metabolite analysis
For the determination of plasma free fraction 200 µL of plasma spiked with formulated [ 18 F]ACI-12589 (300 -500 kBq) were pipetted in duplicates into ultrafiltration units (Amicon Centrifree 30, Millipore) and centrifuged for 20 min at 3000 x g (4400 rpm).The radioactivity of the ultrafiltrate and the filtration unit were measured (dose calibrator, Capintec 120-CRC), and the plasma free fraction could be calculated.b-Amyloid and Tau staining of the AD donor with mixed a-syn pathology (Fig. 2c) Immunofluorescence for Ab (using mAb, 4G8) and Tau (using mAb, MC1) pathology in AD tissue donor sections also used in the data presented in Fig. 2c.Scale bar in top panels, 200 µm.Scale bar in bottom panels, 100 µm.Suppl.

Supplementary Table 1
18 F]ACI-12589 [ 18 F]Fluoride was produced by a cyclotron (GE PETtrace 800) by irradiating [ 18 O]H2O via a (p,n) reaction.The [ 18 F]fluoride (aq.) was transferred to a hot cell containing a Neptis Perform synthesis module (Optimized Radiochemical Applications) for cassette based automated radiosynthesis.The [ 18 F]fluoride was trapped on a quaternary methyl ammonium (QMA) cartridge (K-922, ABX, CO3 2-preconditioned) and [ 18 O]H2O was removed.The activity was eluted into the reaction vial using 1.0 mL of Bu4NH2PO4 (20 mM) dissolved in 50% MeCN in H2O.The eluted mixture was dried via azeotropic distillation by heating the reaction vial to 110 °C under N2-flow with 3 additions of MeCN over 15 min, until the reactor was dried.The chemical precursor ACI-15051, dissolved in dry DMSO was added to the dried [ 18 F]fluoride mixture and the reaction was heated for 10 min.The reaction was cooled and then quenched by addition of 6 mL of H2O.The diluted reaction crude was injected on a semi-preparative HPLC column (Agilent Zorbax eclipse C18, 250 x 9,4 mm, 5 µm, equipped with a 4,6 x 12,5 mm guard column) with an eluent consisting of 25% MeCN in 20 mM citrate buffer (pH 4.4) at a flow rate of 6 ml/min.The fraction corresponding to the desired product was collected for 90 s and diluted by ca 65 mL H2O.The diluted fraction was extracted using a C18-cartridge (Sep Pak C18 light, Waters) previously preconditioned with 5 mL EtOH and washed with 5 mL H2O Arterial blood samples were obtained manually at baseline and 45 seconds (s), 90 s, 135 s, 180 s, 225 s, 270 s*, 315 s, 6 min*, 8 min, 10 min*, 15 min, 20 min*, 25 min, 30 min*, 45 min, 60 min*, 75 min, 90 min* post injection (2 or 5 mL* per sample).Plasma was separated by centrifugation at 3000 x g (4400 rpm; Centrifuge 5702, Eppendorf) for 10 min, and radioactivity was measured in a g-counter (Hidex AMG, energy window 370-1100 keV, calibration constant 0.52 Bq -1 and counting time 30 sec) for both whole-blood and plasma samples (200 µL each).

Suppl. Figure 2 . 5 .
Autoradiography and saturation binding experiments.[ 3H]ACI-12589 and [ 18 F]ACI-12589 display specific binding in human brain tissues containing a-synuclein ex vivo.a) Saturation binding studies with [ 3 H]ACI-12589 on human brain tissue sections from from a familial PD (PD SNCA, top) and a MSA case (bottom).Left, autoradiographic detection of [ 3 H]ACI-12589 binding in brain tissue sections.Total: total binding (2.5 nM -80 nM); NSB: Non-specific binding, as defined by residual binding in the presence of 2 or 5 μM unlabelled ACI-12589.Right, total and non-specific binding of [ 3 H]ACI-12589 with increasing concentration of ligand on the x-axis.Fitting the data was performed with non-linear regression analysis, using a one-site specific binding model in GraphPad Prism.Staining of adjacent sections with a-syn-pS129.PD SNCA: PD with SNCA G51D mutation; MSA: multiple system atrophy; Scale bar, 2 mm.(b) Autoradiography with [ 3 H]ACI-12589 in MSA tissue from the cerebellum brain region and brain region-matched control tissue.Total: total binding (10 nM); NSB: Non-specific binding (5 µM), as defined by residual b binding [³H]ACI-12589 (nM) binding in the presence of 5 μM unlabelled ACI-12589.Immunofluorescence with a-syn-pS129 antibody (top panels) on the same sections.CBM: Cerebellum; Scale bar, 2 mm.Suppl.Figure 4. Confirmation of the presence of pathological b-Amyloid and Tau in tissue donors in Fig. 2a, c. a) Biochemical characterization of AD brain homogenates used to measure specific binding in Fig. 2a, head-to-head with healthy control homogenates and PBS.b-Amyloid and pathological tau levels were measured by AlphaLisa.RU: relative units.Immunofluorescence with a-syn-pS129 antibody or pTau-AT8 antibody in brain tissue sections from two PSP cases showing a-syn pathology (bottom left panels) or tau pathology (bottom right panels).High-resolution ARG with [ 3 H]ACI-12589 (60nM) in the same sections, showing co-labeling of a-syn aggregates (top left panels, red arrows) and absence of binding to tau aggregates (top right panels).Red rectangle in the zoomed-out image (scale bar, 100 µm) indicates the region of magnification for the zoomed-in image (scale bar, 20µm).Suppl.Figure 6.TDP-43 autoradiography.Assessment of target engagement of [ 3 H]ACI-12589 on FTLD TDP-43 type C tissue containing pathological TDP-43 aggregates.High-resolution ARG with [ 3 H]ACI-12589 (60nM) on FTLD TDP-43 type C tissue and PD tissue.Immunofluorescence staining with phospho-TDP-43 (pTDP43) antibody on the same FTLD TDP-43 type C tissue labelling TDP-43 aggregates or with a-syn-pS129 antibody on the PD section, labeling a-syn aggregates.No accumulation of silver grains on TDP-43 aggregates with [ 3 H]ACI-12589.MFG: Middle Frontal Gyrus, AMG: Amygdala; Scale bar, 20 μm.

Time activity curves and
parent fractions.a) Time activity curves for all participating groups in cerebellar white matter (Cer WM), pons, pallidum, putamen, cerebellar grey matter (Cer GM) and occipital cortex (Occ Ctx).b) parent fractions in PD and DLB participants.Ctrl n = 7, DLB n = 2, MSA-C n = 6, MSA-P n = 2, PD n = 5.Suppl.Figure 11.Transversal [ 18 F]ACI-12589 images at the level of the basal ganglia.Shown are 8 controls, 7 PD patients, one DLB patient, the 13 MSA patients and 3 participants with PSP.* indicates that the participant has been taking MAO-B inhibitors (Sel -Selegiline; Saf -Safinamide; Ras -Rasagiline).DLB: Dementia with lewy bodies; MSA-C: Multiple system atrophy with a cerebellar phenotype; MSA-P: Multiple system atrophy with a parkinsonian phenotype; PD: Parkinson's Disease; PSP: Progressive supranuclear palsy; SUVR: Standardized uptake value ratio.Transversal [ 18 F]ACI-12589 images at the level of the middle cerebellar peduncles.Shown are 8 controls, 7 PD patients, one DLB patient, the 13 MSA patients and 3 participants with PSP.* indicates that the participant has been taking MAO-B inhibitors (Sel -Selegiline; Saf -Safinamide; Ras -Rasagiline).DLB: Dementia with lewy bodies; MSA-C: Multiple system atrophy with a cerebellar phenotype; MSA-P: Multiple system atrophy with a parkinsonian phenotype; PD: Parkinson's Disease; PSP: Progressive supranuclear palsy; SUVR:Standardized uptake value ratio.Regional retention of [ 18 F]ACI-12589 in the different diagnostic groups.Graphs in panel a)g) shows data with an occipital cortex reference, panel h) shows the occipital cortex using a cerebellar grey matter reference region.a) middle cerebellar peduncle retention.b) retention in the lentiform nuclei (bilateral putamen and globus pallidus).Retention in the c) pons, d) midbrain, e) thalamus, f) amygdala and g) temporal lobe cortex.h) shows SUVRs in the occipital cortex using a cerebellar grey matter reference region.Boxplots show median, IQR (box) and whiskers (Q1 -1.5*IQR / Q3 + 1.5*IQR or minimum/maximum value, outliers not included).Ctrl n = 8, DLB n = 2, MSA-C n = 8, MSA-P n = 5, PD n = 8, Ataxia n = 3, PSP n = 3, AD n = 5.AD: Alzheimer's Disease; Ataxia: cerebellar ataxias (Friedreich Ataxia and cerebellar ataxia due to a SAMD9L mutation); Ctrl: control subjects; MSA-C: Multiple system atrophy with a cerebellar phenotype; MSA-P: Multiple system atrophy with a parkinsonian phenotype; PSP: Progressive supranuclear palsy; SUVRcer: SUVR with a cerebellar grey matter reference region; SUVRocc: SUVR with an occipital cortex reference region; SUVR: Standardized uptake value ratio.Retention in participants withduplication of the SNCA gene.Transversal [ 18 F]ACI-12589 images at the level of the middle cerebellar peduncles (left row), midbrain (middle row) and the basal ganglia (right row).PD: Parkinson's Disease; SUVR: Standardized uptake value ratio.